Cetaceans comprise a fascinating order of mammals, with many unique morphological features that distinguish them from the rest of the class. Many of these features reflect an extreme adaptation to the aquatic environment, including nasal passages (Kukenthal, 1893; Klima, 1999),absence of hind limbs (Kukenthal, 1895; Sedmera et al., 1997a), polyphalangy of forelimbs (Kukenthal, 1893; Sedmera et al., 1997b), and crocodilian-like dentition (Kukenthal, 1893; Misek et al., 1996), just to name a few.
Little is known about the structure of the cetacean heart. Slijper (1973) analyzed the early literature and noted that, compared to terrestrial mammals, the cetacean heart lies more cranially and horizontally, and in midline within the thorax. A bifid apex has been described in the sperm whale (Physeter macrocephalus), Sirenia, Florida manatee, and dugong. In the sperm whale, it was hypothesized to be an adaptation for deep diving (James et al., 1995); however, this explanation does not hold for the manatee (Caldwell and Caldwell, 1985), which lives in shallow waters and possesses the lowest heart-to-body weight ratio among mammals (Tenney, 1958), or the dugong (Nishiwaki and Marsh, 1985). However, a single apex situated at the level of the fourth intercostal space was described in adult harbor porpoises (Phocaena phocaena) by Slijper (1973).
The bifid apex is normally present during mammalian embryonic development (Sedmera et al., 2000), but most adult mammals have a compact, “typical” heart, in which the apex is formed exclusively by the left ventricle. The origin of the bifid apex lies in the segmental arrangement of the looped tubular heart, such that the trabeculated portions of the left and right ventricles “balloon” out of the heart loop (Moorman and Lamers, 1994). Later in development, however, the left ventricle becomes dominant, with an ellipsoidal shape, and the right ventricle wraps around it like a swallow's nest (Hutchins et al., 1978). This arrangement coincides with the development of the three-layered spiral structure of the compact myocardium (Jouk et al., 2000), and this tight ventricular coupling is beneficial for heart performance (Yacoub, 1995).
Even less is known about heart development in Cetaceans. Bryden (1972) described pre- and postnatal growth and development in some species, but recorded only weights for the heart. Therefore, in the present study we attempted to fill in this gap by providing a basic timetable of major morphogenetic events in the spotted dolphin, and drawing a comparison with commonly studied species.
A, artery; AA, aortic arch; Ao, aorta; AoD, descending aorta; AVC, atrioventricular cushion; Co, compact layer of the ventricle; Ct, conotruncus; CtC, conotruncal cushion; DA, ductus arteriosus; endo, endocardium; eso, esophagus; FL, fore limb; IAS, interatrial septum; IVS, interventricular septum; IVC, inferior vena cava; LA, left atrium; LAA, left atrial appendage; LB, left bronchus; LL, left lung; LV, left ventricle; LSH, left sinus horn; LVV, left venous valve; Mi, mitral valve; myo, myocardium; PM, pectinate muscles; Pu, pulmonary trunk; RA, right atrium; RAA, right atrial appendage; RB, right bronchus; RBB, right bundle branch; RL, right lung; RV, right ventricle; RVV, right venous valve; SV, sinus venosus; SVC, superior vena cava; T, trachea; Tr, trabeculae; Tri, tricuspid valve; V, vein; X., vagus nerve.
MATERIALS AND METHODS
We examined macroscopically and/or histologically 19 dolphin embryos and fetuses at different developmental stages (Table 1). The collection (containing a total of 90 embryos and fetuses of the spotted dolphin) was assembled by the Center of Morphology at J.W. Goethe University in Frankfurt am Main, Germany. The embryos were recovered from pregnant females that were accidentally killed during tuna-fishing operations (see Linehan, 1979; Perrin, 1969), and were fixed and stored in 70% ethanol.
Table 1. Total body length and estimated age of the examined specimens*
Total length (mm)
Estimated age (days)
The duration of gestation in this species is 280 days, and the length at birth 950 mm (Sterba et al., 2000).
The embryos were photographed prior to dissection, and the chest cavity was then carefully opened to obtain a view of the heart. The thorax was separated from the rest of the body by two razor-blade cuts perpendicular to the body axis. It was then dehydrated and processed into paraffin. Serial sections were cut at 7-μ thickness in the transverse plane, and alternate sections were mounted on poly-lysine-coated slides for hematoxylin-eosin staining and immunohistochemistry. Examinations of preexisting histological series from the collection were performed for specimens Sa 3 and Sa 29 (transverse), and Sa 34, Sa 39, Sa 46, and Sa 49 (sagittal). Stained sections were mounted in Eukitt archival mounting media and observed under a Leica DMLB microscope (Leica Microsystems, Bannockburn, IL). In addition to bright-field images, a blue excitation/green fluorescence modality was used to study the development of vasculature, since the erythrocytes filling the forming vessels or present in the heart lumen were distinguishable by their strong autofluorescence. Unfortunately, the level of tissue preservation did not permit immunohistochemical analysis or in situ terminal dUTP nick-end labeling (TUNEL) staining, which limited the examination to standard morphological staining. Pictures were taken using a Hamamatsu 3CCD digital color camera (Hamamatsu, Bridgewater, NJ), and processed with Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA). The sections from the largest specimens (>60 days of gestation) were scanned as transparencies at 2,400 ppi using an HP PhotoSmart photo scanner (Hewlett-Packard, Palo Alto, CA). Digital image processing included adjustment of background and color levels, and Unsharp Mask filtering to enhance contrast.
Morphometry was performed on the digital images of four-chamber cross sections. The proportion of the compact myocardium was measured using an unbiased point-counting technique with Analyze 7.1 software (CNSoftware, London, UK), and defined as the number of intercepts on the compact layer divided by the total number of myocardial intercepts. Point counting was performed on a single representative section of each heart (carefully selected and matched four-chamber view through the plane of both atrioventricular valves). For the sagittal series, a section showing the entire ventricle cut through the atrioventricular orifice was used. This approach was based on the known regional differences in the proportion of the compact myocardium within the ventricles, which we studied previously on systematic transverse sections in the chick (Sedmera et al., 1998). Employing a four-chamber or longitudinal view equalizes the differences, and allows one to make meaningful comparisons between stages. The image used for counting had a pixel dimension of 1,024 × 768, and it was covered by a grid of 110 × 146 points. All of the counts were done twice by a single observer (D.S.), and the difference between repeats was consistently <2%. The compact layer thickness was measured at the ventricular midportion as an average from five measurements. All measurements were performed separately for each ventricle.
The total length and estimated gestation age of the specimens analyzed in this study are listed in Table 1. A complete listing of all specimens available in this collection can be found in Sterba et al. (2000).
The earliest specimen available from the collection (Sa 3, 23 days) could be examined only on a transverse serial section of the whole embryo. All four chambers of the heart were present at this stage (Fig. 1). Both superior caval veins were present at the venous end, and drained into the sinus venosus. Atrial septation was just beginning, with the primary interatrial septum present in the atrial roof, and the mesenchymal protrusion continuous with the dorsal mesocardium on the dorsal side (Fig. 1c and d). The pectinate muscles had begun to form in the atrial appendages. The heart was fixed in the contracted state, bringing the cardiac cushions into close contact. Only the two (antero-superior and postero-inferior) atrioventricular cushions were present in the atrioventricular canal, which was a very prominent segment between the atria and the ventricles. There was a wide communication between the left and right ventricles, which were connected essentially in series. Subepicardial mesenchyme was forming in the interventricular grooves, but no signs of angiogenesis or hematopoesis were observed within this cell population. The outflow tract was relatively long, with myocardial mantle and conotruncal cushions containing numerous mesenchymal cells with very little signs of apoptosis.
The heart of the youngest embryo analyzed both macroscopically and in serial sections (Sa 4, 24 days; Fig. 2) was filled with a rich trabecular network, and had a thin compact layer with no coronary vascular development. The ventricles were partially separated by a well-defined muscular interventricular septum. The atrioventricular cushions were not yet fused. The outflow tract was also undivided, and possessed a well-defined muscular sleeve. Both atria were spherical and already contained well-developed pectinate muscles. The interatrial septum was already formed, and both the primum and secundum interatrial foramens were present. The sinus venosus was well developed, receiving blood from both superior and inferior caval veins. Only the right superior vena cava was present. The pulmonary veins were not yet developed. Programmed cell death was predominantly seen as pycnotic nuclei in the outflow tract cushions. Only a few such apoptotic cells were present in the atrioventricular cushions. No signs of vasculogenesis were noted in the epicardium. The heart of specimen Sa 7 (27 days) showed only a moderate advance in morphogenesis. The arrangement of the atria and ventricles was the same, and the shape of the heart in frontal view was almost identical. At the level of the outflow tract, however, the conotruncal cushions had started to fuse, separating the aorta from the pulmonary artery. A large developing coronary vessel was observed in the anterior interventricular sulcus. Zones of programmed cell death (defined by nuclear pycnosis) were present in both the atrioventricular and outflow tract cushions.
More progress in morphogenesis was seen in the heart of Sa 11 (34 days), in which the most significant change was the bulging of the ventricles, giving the apex a bifid appearance (Fig. 3). The interventricular sulcus thus became more prominent, and the ventricles were more clearly separated. Even though the outflow tracts were now separate, there was still a persisting interventricular foramen (Fig. 3d). Nuclear pycnosis suggestive of apoptotic cell death was present in the cardiac cushions at the mesenchyme–myocardium border. The His bundle and bundle branches were easily recognizable by histological criteria (i.e., larger size of the nuclei, different cell orientation, and stronger eosin staining). The atrial appendages became more prominent. Two pulmonary veins were found draining into the left atrium. In the subepicardium, blood islands and vessels were forming, but no sinusoids were found in the compact myocardium, which was still very thin. At a length of 38 mm (Sa 14, 35 days), ventricular septation was completed. The two ventricles were almost spherical in shape, and very similar in size. The atria were well developed, with their prominent appendages almost completely filled by an extensive network of pectinate muscles. The right sinus horn was now completely incorporated into the right atrium, while the left sinus horn was reduced to a horizontal tunnel, but did not communicate yet with the forming coronary veins. The cusps of the semilunar valves were starting to form in the outflow tract. Very few pycnotic nuclei were seen at this stage. The subepicardium contained numerous blood sinusoids, but none were yet present in the compact myocardium.
In the heart of Sa 28 (39 days), the outflow tract was now well separated externally into the aorta and the pulmonary artery (Fig. 4), and was straighter than before (compare with Figs. 1 and 2). In the atria, the pectinate muscles became more prominent in the appendages. There were now four pulmonary veins entering the left atrium. The ventricles were separated externally by a deep sulcus, and on the inside by a thick interventricular septum. Dorsally, the sulcus was filled in by a wedge of subepicardial connective tissue containing developing coronary vessels. The atrioventricular valve leaflets were now better defined, but the papillary muscles supporting them still consisted of loose trabecular struts. The cusps of the semilunar valves were also well defined. The branches of the ventricular conduction system were beginning to display some fibrous insulation from the surrounding myocardium. The heart of specimen Sa 29 (available in histological series) looked almost identical, including the quantitative parameters. Specimen Sa 34 (40 days), examined in sagittal serial sections, also displayed a similar level of morphogenesis.
In the next specimen (Sa 36, 41 days), the interventricular sulcus was very prominent. The atria had grown in size, and their smooth part began to prevail over the appendages with pectinate muscles. The entrance of the inferior vena cava into the right atrium was guarded by the venous valves (Fig. 5b). Only one (right) superior vena cava entered the right atrium. The remnants of the left sinus horn were still present. In the left atrium, the pulmonary veins were seen to enter on the posterior aspect (Fig. 5c). The atrioventricular valves still displayed thick, immature leaflets, but the papillary muscles had become much better organized (Fig. 5b). The rest of the trabeculae had also thickened, but the compact myocardium remained thin and devoid of coronary vasculature, despite the presence of numerous subepicardial sinusoids. The branches of the conduction systems clearly had a bundle-like appearance, and cells with pycnotic nuclei were often present at their borders. The heart of Sa 39 (42 days, examined on sagittal sections) was very similar, including the lack of an aorto-coronary connection.
The heart of Sa 44 (48 days) had a more compact shape, although the prominent interventricular sulcus still gave the apex a bifid appearance. The trabeculae had thickened considerably (Fig. 6). Clearly defined larger vessels appeared in the subepicardial space, and sinusoids were present in the compact myocardium. The left sinus horn was now fully converted to the coronary sinus, and a valve guarded its entrance to the right atrium. The coronary arteries were connected to the aorta, indicating a functional coronary circulation. The timing of this event was confirmed in similarly aged specimen (Sa 46), examined on sagittal sections. The next specimens available in the collection (Sa 49, 51 days) showed moderate autololytic changes. The most prominent feature was now a clear difference between the trabecular patterns and the compact layer thickness between the left and right ventricles. The shape of the apex was difficult to ascertain in this orientation. The heart of Sa 54 (60 days) was not well preserved: it presented bloody effusion throughout the pericardial cavity, and fragmentation of the myocardium due to postmortem development of air bubbles. However, we were able to ascertain that the heart was clearly more compact than in the previous specimens, but the apex was still bifid. The trabeculae were thick, and arranged along the ventricular perimeter at regular intervals. A fine trabecular network was present in the apical regions. The compact myocardium was notably thickened, and supplied by coronary vessels.
The well-preserved heart of Sa 60 (67 days) showed a compact shape and advanced fetal morphology. The apex was single and formed entirely by the left ventricle, yet the interventricular groove was still very prominent. The coronary blood supply now encompassed both the substantial compact layer and trabeculae. The arrangement of the chambers was the same as in the adult, and the valves had an almost mature morphology. The atria were large, with thin walls, and the venous connections showed the definitive arrangement (four pulmonary veins draining to the left atrium, superior and inferior caval veins, and coronary sinus entering the right atrium). Well-developed internal thoracic vessels and phrenic nerves were observed adjacent to the parietal pericardium (Fig. 7).
The last specimen studied histologically (Sa 73, 85 days) was not well preserved. Externally, the heart had a compact shape. The apex was single and was formed by both ventricles (Fig. 8). Coronary vessels were seen running on the surface in a typical pattern, and the perfusion extended all the way through the myocardium to the trabeculae. No further details could be obtained because of extensive tissue shredding due to air bubbles. Macroscopic analysis of older hearts confirmed that the heart shaped remained the same, with a prominent anterior descending branch of the left coronary artery in the interventricular groove (Fig. 8), and a single, pointed apex formed entirely by the left ventricle.
The proportion of the compact myocardium remained within the 30–40% range for a lengthy period. In the younger specimens, the right ventricle appeared to have a higher proportion than the left one, but then the trend reversed (Fig. 9). A distinct increase was noted from day 60, and the left ventricle was the first to gain substantially. Measurements of compact layer thickness confirmed our impression that the compact layer remained thin for a long period, correlating with lack of coronary perfusion. An increase in left ventricular thickness was observed after the coronary circulation was established (day 48). A significant increase in thickness in both ventricles was observed in the most advanced specimens (>60 days old), and the left-to-right ratio then was about 2:1.
According to the approximate length of gestation in dolphins of 280 days (Sterba et al., 2000), the developmental time points in the spotted dolphin would best compare with humans, for which extensive collections of embryonic material exist (O'Rahilly, 1979; Thompson et al., 1985). In the embryonic human heart, the temporary presence of a bifid apex was noted in embryos of crown–rump length 17–37 mm by Mall (1912). A similar situation is found in other mammals (Pexieder et al., 1984; Vuillemin and Pexieder, 1989a) at comparable developmental stages. In birds, however, the apex is single and formed by the left ventricle only in the embryonic stage (Ben-Shachar et al., 1985; Sedmera et al., 1997c). The persistence of the bifid apex into adulthood has been described in Sirenians (Caldwell and Caldwell, 1985; Tenney, 1958; Nishiwaki and Marsh, 1985) and sperm whales (James et al., 1995). However, it does not appear to be a universal feature among larger aquatic mammals, as shown in a study of harbor porpoises (Slijper, 1973) and in the present work. It has been suggested that loose interventricular coupling with the bifid apex may be an adaptive mechanism for diving, to facilitate more independent blood pumping (James et al., 1995). However, there are considerable differences in physiological adaptations for diving among marine mammals, such as seals and dolphins (Harrison and King, 1980). From a mechanical point of view, the absence of interventricular coupling is disadvantageous for right ventricular pumping function, which is normally aided by the left ventricle (Yacoub, 1995). This may be tolerable for an animal with large functional reserves or a sedentary lifestyle (e.g., the manatee), but not for active, long-distance swimmers like most marine dolphin species.
Cardiac septation is completed at 8 weeks in humans (O'Rahilly, 1979; Pexieder and Janecek, 1984), 8 days in the chick (Ben-Shachar et al., 1985; de la Cruz et al., 1997), and ED14 in the mouse (Vuillemin and Pexieder, 1989b). We found that the interventricular foramen closed at about 35 days in the spotted dolphin, and by that time the septation of the atrioventricular canal and the conotruncus was complete. This is slightly earlier than in humans; however, the other comparable features of the embryo were at a similar developmental level (Sterba et al., 2000). Similar to the situation in humans (Thompson et al., 1985), the conotruncus was initially bayonet-shaped, and straightened at about the same time in development (35 days). The only previous histological study of embryonic Cetaceans (Stenella coeruleoalba, 12-mm crown–rump length (Hosokawa, 1955)) described the heart just prior to completion of outflow tract septation, and the description corresponds well with our findings in specimens of a similar stage (Sa 11 and Sa 14, with a crown–rump length 15 and 12 mm, respectively).
We observed four pulmonary veins finally entering the left atrium, similarly to the situation in humans. The number of pulmonary veins (reflecting the degree of absorption of the pulmonary confluence to the left atrium) varies among mammals, ranging from a single pulmonary vein in the mouse (Webb et al., 1998) to four to six in Atriodactyla, which are currently considered the closest relatives of Cetaceans (Milinkovitch et al., 1993). The presence of pectinate muscles in the atria of even the earliest available specimens confirms that cardiac morphogenesis was already well advanced, since the pectinate muscles first appear in stage-27 chick, ED12 mouse, or Carnegie stage-16 human hearts (reviewed in Sedmera et al., 2000).
Apoptotic cell death plays a major role in heart morphogenesis (Pexieder, 1972). In accord with Pexieder's (1972) classic description, we found prominent areas of cell death in the developing cardiac cushions, particularly in the conotruncal region. Occasional apoptotic cells were also observed in association with developing bundles of the conduction system, in agreement with our previous observations in the chick embryo (Cheng et al., 2002). As in that chick study, apoptosis in the outflow tract cushions appeared to precede apoptosis in the atrioventricular canal cushions.
One prominent internal feature of the preseptation mammalian heart that begins to function very early in development (Rentschler et al., 2001) is the ventricular conduction system. This system was identified morphologically in ED 11 mouse heart by Viragh and Challice (1977). Immunohistochemical studies showed the precursors of the conduction system in the human heart at 7 weeks (Wessels et al., 1996). While the bundles can not be demonstrated histologically before the completion of ventricular septation (day 8) in the chick (Vassall-Adams, 1982), immunohistochemical markers (HNK-1, PSA-NCAM) are able to detect the precursors at preseptation stages (Chuck and Watanabe, 1997). In the spotted dolphin, we were able to identify the bundle branches prior to completion of ventricular septation at 35 days, but their insulation was not completed until more advanced stages.
Coronary vessels develop from the subepicardial mesenchyme by the process of vasculogenesis (reviewed by de la Cruz et al., 1999). First stages (blood islands and primitive unconnected vessels) are observed at early preseptation stages (4 days) in the chick (Hiruma and Hirakow, 1989; Poelmann et al., 1993). However, the connection between the coronary arteries and the aorta is not established until the completion of septation (ED7-8), and apoptosis plays an important role in the penetration of the aortic wall (Velkey and Bernanke, 2001). In rats, coronary vasculogenesis follows a similar pattern (Ratajska and Fiejka, 1999). In humans, the first signs of coronary vasculogenesis in the subepicardium have been observed at 28–30 days (Hirakow, 1983; Conte and Pellegrini, 1984). However, it is not known when exactly the connection with the aorta is established. In the present study, we observed the first signs of vessel formation at 27 days, but connections with the aorta were not seen until 48 days. The establishment of aorto-coronary connections corresponded with the increase in proportion and thickness of the ventricular compact myocardium.
The compact layer of the ventricle is initially very thin (reviewed in Sedmera et al., 2000), and trabeculae form most of the myocardial mass at preseptation stages (Blausen et al., 1990; Sedmera et al., 1998). The proportion of the compact myocardium correlates well with its thickness, and increases in chick and mouse around the time of completion of ventricular septation (Sedmera et al., 2000). This sudden rise in compact layer thickness, which is more pronounced in the left ventricle, is caused by trabecular compaction, and necessitates coronary perfusion to provide an adequate supply of nutrients and oxygen to the myocardium. The values of compact layer proportion and thickness found in these few dolphin specimens (Fig. 9) compare well with the values reported for other species at comparable stages (Blausen et al., 1990; Sedmera et al., 1998). The relatively long time between the completion of ventricular septation (35 days) and trabecular compaction resulting in increased compact layer thickness (60 days) was surprising; however, the correlation between the relatively sudden onset of compaction (Sedmera et al., 2000) and coronary perfusion (Poelmann et al., 1993) was maintained. A similar delay in morphogenesis was recently reported in other dolphin organ systems (i.e., ossification and odontogenesis) by Sterba et al. (2000).
Unfortunately, no embryos younger than 22 days of gestation were available for analysis (this relates to the way in which the material was obtained—the small embryos were most likely missed by the fishermen). The quality of preservation of this material did not permit us to use any immunohistochemical or in situ techniques, so we were limited to standard histological staining. Together with the inherent uncertainty as to the age of the embryos, the small number of specimens available for each age group did not permit extensive quantitative comparisons, apart from longitudinal trend measurements. As we noted previously (Sedmera et al., 1997b), it is impossible to obtain additional fresh material because Cetaceans are protected.
We thank Ms. Mauricette Capt (University of Lausanne, Switzerland), Ms. Jarmila Fullerova (Charles University, Prague), and Ms. Jana Kriva (Academy of Sciences of the Czech Republic, Brno) for tissue sectioning, and Dr. Andy Wessels (Charleston, SC) for a critical reading of the manuscript.